Abstract Craniofacial abnormalities affecting the mandible, maxilla and jaw joint are commonly encountered birth defects, most of which require surgical correction to establish quality of life and in some cases survival. While one or more organizing centers within the developing pharyngeal arches, from which the face arises, may hold the key to understanding the etiologies of these defects, the existence of such centers has never been proven, leading to a poor understanding of how gene regulatory networks are regulated and integrated during facial development. This limiting knowledge stifles new approaches to efficaciously treat these deformities. Endothelin1 (through DLX proteins) and BMP (through SMAD proteins) signaling establishes positional and structural identity of neural crest cells within the ventral mandibular arch. This is primarily achieved by induction of the basic bHLH transcription factor HAND2. HAND2 and BMP synergy is required for induction of the bHLH factor HAND1 within the ventral-most ?cap? of the mandibular arch. Interestingly, altering HAND1 dimer partner choice (thus altering HAND1-mediated signaling) results in pronounced mid-facial clefting, even though Hand1 is not expressed in the mid-face structures. Our data shows that loss of either BMP or HAND2 activity disrupts the establishment of the ventral cap. These findings establish our hypothesis that the intersection of BMP and HAND2 activity establishes a ventral cap-signaling center, which acts in both cell and non-cell autonomous manners to drive upper and lower jaw development. Additionally, we hypothesize that DLX activity antagonizes BMP/HAND2 synergy. This proposal takes advantage of the craniofacial development expertise of Dr. David Clouthier, the bHLH signaling expertise of Dr. Anthony Firulli and a number of novel mutant mouse alleles to test these hypotheses in two Aims. In Aim 1, we will use single cell (sc) RNA-seq to define the gene regulatory networks that are initiated by the coordinated action of both HAND2 and BMP that act either in an autonomous (ventral cap) or non-cell autonomously (more dorsal first arch areas) manner. Following analysis of scRNA-seq data, the top HAND2/BMP effector candidates will be functionally evaluated in loss-of-function and genetic studies. In Aim 2, the role of DLX proteins in confining ventral cap size will be examined using a novel gain-of- function Dlx5 mouse allele. Chromatin accessibility influenced by DLX proteins will also be examined using ATAC-seq, followed by functional testing of DLX action by creating a Hand1 mouse mutant lacking DLX cis- element inputs. Together, these novel approaches will provide the first direct evidence that the mandibular arch ventral cap is a signaling center required for facial development. Relevance: Craniofacial abnormalities are common and require intensive reconstructive surgical corrections. HAND2 and BMPs play key roles in patterning the neural crest cells that form the face. Gaining insight into the molecular mechanism of this understudied developmental process could have great potential for initial development of non-surgical treatments for congenital craniofacial defects in patients.